Chromium carcinogenesis: Characterization of DNA damaging intermediates by EPR phosphorus-31 NMR, HPLC, ESI-MS and magnetic susceptibility

The hydrolytic cleavage and oxidative degradation mechanisms of dGDP by the oxochromate-(V) complexes bis(2-ethyl-2-hydroxybutanoato)oxochromate(V) (I) and bis(hydroxyethyl)-amino-tris((hydroxymethyl)methane)oxochromate(V) (II) in the presence of H2O2 were investigated at neutral pH. The products of the reactions were separated and characterized by chromatographic and spectroscopic methods. The oxidative degradation is supported by the detection of free G, furfural, phosphoglycolate, pyrophosphate, guaninepropenal, 8OHdG and guanidinohydantoin. These products are formed through two parallel mechanisms that start with a common Cr-dGDP intermediate in which the oxochromate binds the alpha phosphate moiety followed by abstraction of H from C4' and C5' from the ribose. The detection of inorganic phosphate and dGMP suggests that when the oxometal binds the beta phosphate it mainly promotes hydrolytic cleavage of the phosphate diester bond. By estimating the amount of each catabolite it was concluded that the oxo metal ion does not show selectivity during the hydrogen abstraction and that oxidation of the substrate is preferred over its hydrolysis.;The reaction between diperoxoaquaethylendiamine chromium(IV) ( III) and glutathione (GSH) at neutral pH was studied by EPR and ESI-MS. The ions of m/z ratios of 450 and 757 were identified as intermediates while the ions of m/z ratios of 484 and 775 were identified as products. Three EPR signals detected at g = 1.996, 1,986 and 1.983 were attributed to Cr(V) intermediates while a signal at g = 1.975 (peak to peak line width = 259.72 G) that appeared after the Cr(V) signals had disappeared was attributed to Cr(IV). Spin trapping experiments with DMPO and DEPMPO revealed that the GS radical but not OH radical was formed during this reaction. The GS radical and the Cr(V) intermediates detected by EPR confirmed that the reaction occurs in a series of one electron transactions. Overall this reaction proceeds in cycles. The reaction produces Cr(V) intermediates followed by accumulation of a chromium(IV) second intermediate that decays slowly to a Cr(III) product. The DNA damage assay suggests that during its reaction with glutathione, III becomes a DNA damaging agent.;The mixture of intermediates of the reaction between chromate and glutathione in glycine buffer (pH 2.8) was investigated with a SQUID susceptomer. The saturation magnetic moment was calculated to be 1.39 muB while the effective magnetic moment was 2.55 muB. Based on the saturation magnetic moment, the intermediates were characterized as 30% Cr(IV) and 69% Cr(V). Using the effective magnetic moment the mixture was characterized as 58% Cr(IV) and 42% Cr(V). The spin only formula gave a total spin angular momentum of 0.88 which supports Cr(IV) as the predominant intermediate. As previous studies did not detect Cr(V) EPR signals, it was assumed that only a Cr(IV) intermediate was formed. In light of the present work and stoichiometric data from previous work, the silent part of the mixture of intermediates was interpreted as a Cr(V) dimer in which the Cr centers are diamagnetically coupled. With this data in consideration, the proportion of Cr(IV) in the mixture of intermediates should be at least 65%.